Exposure to reactive oxygen species is an inevitable consequence of aerobic life. Damage from reactive oxygen species, termed oxidative stress, has been associated with several human disease states, including cancer, cardiovascular disease, and neurodegenerative disease. In addition, damage from oxygen radicals has been implicated in the normal aging process. Therefore, understanding how cells are protected from oxidative stress is an important health-related issue. The baker's yeast Saccharomyces cerevisiae provides an excellent model system in which to study the basic cellular mechanisms of oxidative stress protection. One critical line of defense against oxidative stress is provided by the pentose phosphate pathway, a metabolic pathway that provides a source of cellular NADPH. A key pentose phosphate pathway enzyme is glucose 6-phosphate dehydrogenase, which is encoded by the ZWF1 gene in yeast. Although ZWF1 is known to be involved in protecting cells from oxidative damage, the precise role of ZWF1 and related factors in the oxidative stress protection system is not fully understood. The broad, long-term objective of this proposal is to investigate the role of ZWF1 and related yeast genes in oxidative stress protection, to better understand this defense system. A genetic approach will be taken to achieve this objective. Genetic suppressors of the zwf1delta mutation have been isolated in yeast. These suppressors include the mutational suppressor pho85 (PH085 encodes a cyclin-dependent kinase), the multi-copy suppressors ZMS1 and ZMS2 (ZMS1 and ZMS2 encode zinc-finger proteins of unknown function), and two unidentified multi-copy suppressors. Specific Aim I will focus on determining the mechanism by which a pho85 mutation suppresses zwf1delta. Specific Aim II will focus on determining the mechanism by which overexpression of the ZMS1 and ZMS2 genes suppress zwf1delta. Specific Aim III will focus on characterizing novel multi-copy suppressors of zwf1delta. The experiments proposed to carry out the specific aims involve techniques of molecular biology and molecular genetics that can be readily performed by undergraduate students. These experiments are expected to yield insight into how eukaryotic cells are protected from oxidative stress by discovering more about how ZWF1 and related yeast genes protect cells from oxidative damage.